Genome scale molecular analysis has revolutionized the experimental process by which the genes and pathways that influence the initiation, progression, and treatment of cancer can be identified. This has opened up many exciting avenues of biomedical research, but it has also served to confirm the genetic and cellular complexity that underlies tumorigenesis. The identification of the gene silencing mechanism, RNA interference (RNAi), initially in invertebrates and later in mammalian cells, has had enormous implications for our understanding of the regulation of gene expression and our ability to modulate it experimentally. This project is focused on the hypothesis that molecular and phenotypic perturbations induced by RNAi will give insight into the biology of cancer and identify novel anti-cancer molecular targets. The intention of most studies exploiting the RNAi mechanism for loss of function (LOF) analysis is the gene-specific cleavage of protein encoding mRNAs. Technologies that exploit the endogenous RNA-based gene silencing mechanism, RNAi, have developed rapidly for the dissection of gene-function relationships and as a means of furthering molecular target analysis. The overall goal of this project is the enhanced application of RNAi-based technologies for the study of cancer biology. To do this we are focusing on establishing protocols and assays for the reproducible assessment of the effects of RNAi at a molecular and functional level that can be used in mammalian cell line model systems appropriate for the study of tumorigenesis. We are applying optimized and robust protocols for inducing RNAi in cells using synthetic siRNAs and other RNAi effectors, such as short hairpin RNAs (shRNAs) and the use of quantitative assays for analyzing the efficacy of RNA. To date we have examined the silencing mediated by RNAi effectors corresponding to several hundred human genes. Following the induction of RNAi we are applying methods that can assay multiple molecular and phenotypic end-points to assess the function of a protein within specific cancer related pathways and at a broader systems level. Methods we employ include multiplex mRNA assays that allow us to study the downstream effects of LOF on specific pathways and whole transcriptome expression profiling (microarray based analysis or RNA-Seq) that enable us to identify new roles for genes in an unbiased manner. To support this work we have begun to develop and assess bioinformatic tools that can help us to identify non-targeted, but sequence-specific, affects on gene expression following introduction an RNAi effector into a cell. Our molecular based assays are coupled with functional studies chosen on the basis of the putative or defined role of the specific protein under study. During FY14 we have been involved in studies that are using RNAi based technologies to (1) study genes required for the survival of ovarian cancer cells, (2) investigate genes associated with susceptibility to prostate cancer, and (3) identify genes required for the survival of pediatric sarcoma. Finally, this project continues to be critical for assisting and training CCR Investigators in the application of RNAi analysis, and for the establishment of protocols for RNAi screens targeting thousands of human genes (see Z01 BC 010615).